Metabolomics is emerging as an important approach to study biological systems. Measurement of many metabolites at one time has led to important discoveries of disease biomarkers, drug effects, and mechanism of cellular function. Despite the success of metabolomics, current analytical methods are limited in their ability to measure a large fraction of the metabolome. As a result, many changes in the metabolome are not detected by current techniques. High pressure liquid chromatography coupled to mass spectrometry (HPLC-MS) is a prominent method for metabolomics. In this work, we seek to develop new approaches to HPLC that will vastly improve the resolving power for complex mixtures and apply these methods to metabolomics. The resolving power of HPLC increases with column length and with decreasing diameter of stationary phase particles. Changing these dimensions of the HPLC column also increases the pressure required to pump mobile phase through the column. HPLC has traditionally been limited to about 8,000 psi (lbs/in2), but advanced commercial systems can generate up to 19,000 psi. Application of these ultra high pressure LC (UHPLC) systems has yielded significant improvement in the ability to detect metabolites in complex mixtures; however, these systems still fall far short of the goal of resolving all metabolites in common biological samples. We will develop UHPLC systems with capability of 100,000 psi and compatible LC columns. We will investigate use of both reversed phase and hydrophilic interaction liquid chromatography at these extreme pressures to measure metabolites across a broad polarity range. We hypothesize that the new technology will allow measurement of thousands of more metabolites in a given sample than currently possible. The new method should also enhance sensitivity and reproducibility of metabolite detection in complex samples. The new methods will be used to identify metabolic pathways involved in models of adipogenesis, adaptation to high exercise capacity, and diabetic complications. Further, newly developed instrumentation and methods will be incorporated into a national center for metabolomics to ensure widespread impact of the new technology.